Parkinson's disease (PD) is a neurodegenerative disorder that is characterised by the loss of dopaminergic neurons in the substantia nigra. This ultimately leads to dopamine depletion in the striatum causing severe motor deficits. One treatment of Parkinson's disease is the oral administration of L-DOPA, the precursor to dopamine, which can restore a degree of the motor function. However, as the disease progresses, L-DOPA therapy becomes less effective in the treatment of the motor deficits, requiring higher doses to be used which have severe side effects.
Improved treatment of PD could be achieved by the release of dopamine directly to the striatum. This may be achieved by gene therapy. Gene therapy is attractive for the treatment of PD because specific protein production can be targeted to specific areas of the CNS such as the striatum. Synthesis of dopamine from the amino acid tyrosine involves the enzymes tyrosine hydroxylase (TH), which catalyses the synthesis of L-DOPA from tyrosine and aromatic amino acid decarboxylase (AADC), which converts L-DOPA to dopamine. The TH step is thought to be rate limiting. TH requires a cofactor in order to function, tetrahydrobiopterin (BH4), the synthesis of which is catalyzed by the enzyme GTP-cyclohydrolase 1 (CH-1).
Gene therapy approaches using Adeno-associated virus (AAV) vectors to deliver single genes from the dopamine biosynthetic pathway have been investigated with some behavioural benefit in PD animal models (Leff et al., 1999 Neuroscience 92, 185-196; Bankiewicz et al., 2006 Mol Ther 14, 564-570). Further approaches showed that simultaneous delivery of two or more of the enzymes that mediate dopamine synthesis demonstrated greater efficacy in animal models of PD (Fan et al., 1998 Hum Gene Ther 9, 2527-2535.; Shen et al., 2000 Hum Gene Ther 11, 1509-1519.; Muramatsu et al., 2002 Hum Gene Ther 13, 345-354).
This led to the theory that if all three dopamine synthesizing enzymes could be expressed in conjunction and in the same cell then the dopamine synthesis would be greater and would lead to a greater efficacy in PD. However, due to the limited packaging capacity of AAV vectors the number of genes that can be delivered is limited. Thus, it was decided to use a lentiviral vector (LV) for this purpose as the packaging capacity of these vectors is much greater. Lentiviral vectors (LV) are particularly advantageous for gene therapy approaches to the central nervous system (CNS) because of their ability to stably transduce non-dividing cell types such as neurons. LVs derived from non-primate lentiviruses, not known to be infectious or pathogenic for humans, such as equine infectious anaemia virus (EIAV), have been developed and their ability to transduce non-dividing cells established. ProSavin® is an Equine Infectious Anaemia Virus (EIAV) based LV for the treatment of PD. The genome of ProSavin® is a tricistronic construct comprising the coding sequences for the three key dopamine biosynthetic enzymes, TH, AADC and CH1 operably linked by two internal ribosome entry sites (IRES). ProSavin® is currently under evaluation in a phase I/II clinical trial with vector material that was generated in a process comprising three plasmid transient co-transfections of HEK293T cells (Mitrophanous et al., 1999 Gene Ther 6, 1808-1818).
A goal of any gene therapy approach is to increase the titre of the vector, so that lower volumes of vector preparation may be used. This is a particularly desirable outcome in a ProSavin®-type treatment, where the vector is injected directly into the brain, necessitating the use of small volumes.
The complex secondary structures of IRES elements may act as an impediment to efficient reverse transcription. The present inventors therefore hypothesised that removal of the IRES elements should increase the titre of the vectors. One option would be to replace the IRES elements with sequences encoding short peptide sequences (linkers) to generate fusion proteins comprising two or more of the three enzyme activities required for dopamine synthesis. However, as the native form of TH exists as a homotetramer (Goodwill et al., 1997 Nat Struct Biol 4, 578-585) and that of CH1 as a homodecamer (Nar et al., 1995 Structure 3, 459-466; Steinmetz et al., 1998 J Mol Biol 279, 189-199), fusion of these enzymes with each other or with other enzymes such as AADC might prevent the correct tertiary structure formation of the enzymes which may then inhibit enzyme function or prevent them from functioning at maximal capacity. In support of this, it has been previously reported that a fusion between TH and β-galactosidase is enzymatically inactive (Wu and Cepko (1994) J Neurochem 62:863-72).
Surprisingly, the present inventors found that fusion of two or all three of the dopamine synthesising enzymes lead to i) functional enzymes; and ii) an enhanced dopamine biosynthetic pathway for some constructs resulting in elevated levels of L-DOPA and/or dopamine production. In particular, enhanced dopamine production was observed for some constructs when compared to the levels obtained using the construct with all three genes encoding the dopamine synthesising enzymes separated by IRES sequences. Contrary to expectations, for many constructs, the improvement resulting from these fusion designs was not associated with an increase in vector titre indicating that the IRES sequences were not having an inhibitory effect on titre. Furthermore, the increased levels of L-DOPA and dopamine were not due to increases in protein expression from the fusion design vectors suggesting that the fusion design had a higher specific activity.